Project Abstract The primary driver of drug resistance in mycobacterial pathogens like Mycobacterium tuberculosis is genetic mutation, however the molecular processes which govern mutation and mutation avoidance in these organisms remain poorly understood. In nearly all other organisms, mutation rate is tightly controlled by a DNA mismatch repair (MMR) pathway that, immediately after replication, repairs mismatched nucleotides that would become permanent genetic mutations if not corrected and, during senescence, inhibits improper recombination events. Most actinobacteria?which includes mycobacteria?despite having similar basal mutation rates, appear to lack any homologues of the conserved MMR proteins. Rather, it was not until 2017 when it was identified that many actinobacteria instead harbor homologues of archaeal mismatch-sensitive endonucleases Pyrococcus abyssi NucS and Thermococcus kodakarensis EndoMS, and that the native MSMEG_4923 gene product, the ?EndoMS/NucS? (EN) protein, in Mycobacterium smegmatis conferred similar anti-mutagenic and anti-recombination phenotypes that typically define canonical MMR. To date, the mechanisms of EN- coordinated mutation avoidance (ENMA) remain cryptic and poorly understood, and little else is known about the mechanism by which the EN protein would promote mutational avoidance at the molecular level, or even what other proteins are involved in this process. While the ENMA might represent a new opportunity to understand and potentially counter drug resistance and multi-drug resistance (MDR) in mycobacterial pathogens, the absence of fundamental knowledge regarding its mechanism and pathway will limit those opportunities. The long-term goal is therefore to define the mechanism and architecture (components and interactions) of ENMA so that this knowledge can be used to understand and address the challenges of MDR in treating mycobacterial infections. To do so, the purpose of this R21 is to apply a novel assay that is capable of directly characterizing MMR-like activity in living Escherichia coli as an experimental basis for deconstructing the molecular mechanisms of ENMA in living M. smegmatis. The novel assay has many advantages to deconstructing MMR-like activities in mycobacteria that traditional approaches to studying MMR lack, and equipped with this novel biotechnology we will elucidate the foundational mechanisms of ENMA and how it is similar or differs from the canonical MMR reaction. Performing this assay in combination with next-generation biotechnologies like CRISPR, we will also identify and characterize suspected modulators of mycobacterial ENMA or DNA repair-associated toxicity. This unique approach holds the promise of efficiently elucidating the architecture and mechanism of ENMA. This project will then set the foundation for ambitious R01-stage investigation into mechanisms of mutation and drug resistance in mycobacterial pathogens and how it EN may be exploited to provoke mycobacterial cell death.